PROJECT SUMMARY/ABSTRACT Dissemination of multidrug-resistant pathogens has undermined the efficiency of antibiotics and urged a more thorough understanding of bacterial pathogenicity. Bacterial pathogens developed various elegant and sophisticated ways to disrupt and usurp the actin cytoskeleton, which plays numerous vital roles in human defense mechanisms. By hijacking the actin cytoskeleton, pathogenic toxins disturb cell morphology, cell motility, phagocytosis, epithelial permeability, and antigen presentation. Being constantly tuned to the host cytoskeleton by co-evolution, they recognize weaknesses in the host defense and represent powerful tools that foster the understanding of the cytoskeleton on molecular and cellular levels. The long-term goals of the project are to decipher molecular and cellular mechanisms of bacterial toxins targeting the actin cytoskeleton and to utilize the obtained knowledge for understanding functions of the actin cytoskeleton in norm and pathology. The current proposal is directly relevant to the NIH mission as it focuses on two families of related toxins, VopF/VopL and VopM/VopV, produced by human pathogens Vibrio cholerae and Vibrio parahaemolyticus. Both are a common cause of seafood poisoning, while the spread of V. parahaemolyticus has rendered it a major health threat worldwide. Both toxin families are known to affect actin, but their pathogenic mechanisms remain poorly understood. Vop toxins are predicted to cooperate, but the understanding of their synergistic effects is impossible without an in-depth understanding of their individual mechanisms. Research strategy: To assure scientific rigor, two toxins in each family will be characterized in parallel using several highly complementary experimental approaches. Specifically, the effects of the toxins on actin dynamics in bulk and at the single-filament level will be combined with cell biology approaches. Cellular targets of the toxins will be identified by a combination of proximity labeling and mass spectrometry. In Specific Aim 1, the methodological gap between molecular and cellular mechanisms of toxicity will be addressed by live-cell imaging at the single-molecule level to reveal the molecular behavior of VopF/L toxins in host cells. The hypothesis will be tested that uncontrolled multidirectional polymerization of actin by the toxins results in disruption of actin polarity. Specific Aim 2 will reveal novel mechanisms employed by VopM/V toxins. The hypothesis will be tested that hijacking the actin cytoskeleton by VopM/V toxins disrupts the ability of the cell to respond to external and internal stimuli leading to compromised cell integrity. Knowledge gained in the course of the proposal will be applied to discover currently unrecognized elements of the actin cytoskeleton involved in the mechanical homeostasis of the intestinal epithelium. The proposed study is both significant and innovative as it fills a major gap in our understanding of the toxicity of several life-threatening pathogens, reveals novel mechanisms for two families of bacterial toxins, and enables the research team to utilize the acquired knowledge by creating tools for deeper understanding of the actin cytoskeleton.